The present invention relates to glycosyl hydrolase genes for the biotechnological production of oligosaccharides, especially sulfated oligocarrageenans and more particularly oligo-iota-carrageenans and oligo-kappa-carrageenans, by the biodegradation of carrageenans.
The sulfated galactans of Rhodophyceae, such as agars and carrageenans, represent the major polysaccharides of Rhodophyceae and are very widely used as gelling agents or thickeners in various branches of activity, especially agri-foodstuffs. About 6000 tonnes of agars and 22,000 tonnes of carrageenans are extracted annually from red seaweeds for this purpose. Agars are commercially produced by red seaweeds of the genera Gelidium and Gracilaria. Carrageenans, on the other hand, are widely extracted from the genera Chondrus, Gigartina and Eucheuma.
Carrageenans consist of repeat D-galactose units alternately bonded by .beta.1.fwdarw.4 and .alpha.1.fwdarw.3 linkages. Depending on the number and position of sulfate ester groups on the repeat disaccharide of the molecule, carrageenans are thus divided into several different types, namely: kappa-carrageenans, which possess one sulfate ester group, iota-carrageenans, which possess two sulfate ester groups, and lambda-carrageenans, which possess three sulfate ester groups.
The physicochemical properties and the uses of these polysaccharides as gelling agents are based on their capacity to undergo ball-helix conformational transitions as a function of the thermal and ionic environment [Kloareg et al., Oceanography and Marine Biology--An annual review 26: 259-315 (1988)].
Furthermore, carrageenans are structural analogs of the sulfated polysaccharides of the animal extracellular matrix (heparin, chondroitin, keratan, dermatan) and they exhibit biological activities which are related to certain functions of these glycosaminoglycans.
In particular, carrageenans are known:
(i)--for their action on the immune system, causing the secretion of interleukin or prostaglandins, PA1 (ii)--for their antiviral action on the AIDS virus HIV1, the herpes virus HSV1 and the hepatitis A virus, PA1 (iii)--as antagonists of the fixation of the growth factors of human cells, PA1 (iv)--and also for their action on the proliferation of keratinocytes and their action on the contractility of fibroblasts. PA1 1) production of a glycosyl hydrolase by the culture of a marine bacterium; PA1 2) enzymatic hydrolysis of the carrageenan with the glycosyl hydrolase thus obtained; and PA1 3) fractionation and purification of the oligocarrageenans obtained. PA1 RC.sub.1 and RC.sub.2 are the number of hydrophobic residues in the cluster of protein 1 (cluster 1) and the cluster of protein 2 (cluster 2), respectively. PA1 CR is the number of hydrophobic residues in the cluster 1 which correspond to the hydrophobic residues in the cluster 2. PA1 (i)--strongly hydrophobic residues: V, I, L and F; PA1 (ii)--moderately hydrophobic residues: W, M and Y PA1 (iii)--weakly hydrophobic residues: A and C are virtually insensitive to the hydrophobic character of their environment; and PA1 (iv)--hydrophilic residues: D, E, N, Q, H, K and R.
Furthermore, oligocarrageenans act on the adherence, the division and the protein synthesis of human cell cultures, doubtless as structural analogs of the glycosylated part of the proteins of the extracellular matrix. In plants, oligocarrageenans very significantly elicit enzymatic activities which are markers of growth (amylase) or of the phenolic defense metabolism (laminarinase, phenyl-alanineammonium lyase).
Carrageenans are extracted from red seaweeds by conventional processes such as hot aqueous extraction, and oligocarrageenans are obtained from carrageenans by chemical hydrolysis or, preferably, by enzymatic hydrolysis.
The production of oligocarrageenans by enzymatic hydrolysis generally comprises the following steps:
Microorganisms which produce enzymes capable of hydrolyzing iota- and kappa-carrageenans were isolated by Bellion et al. in 1982 [Can. J. Microbiol. 28: 874-80 (1982)]. Some are specific for .kappa.- or .iota.-carrageenan and others are capable of hydrolyzing both substrates. Another group of bacteria capable of degrading carrageenans was characterized by Sarwar et al. in 1983 [J. Gen. Appl. Microbiol. 29: 145-55 (1983)]. These yellow-orange bacteria are assigned to the Cytophaga group of bacteria and some of these bacteria have the property of hydrolyzing both agar and carrageenans.
Purification and characterisation of several .iota.-carrageenases and .kappa.-carrageenases, such as the .iota.-carrageenase and .kappa.-carrageenase of Cytophaga drobachiensis, the .iota.-carrageenase of Alteromonas fortis and the .kappa.-carrageenase of Alteromonas carrageenovora, were described in the thesis of P. Potin ["Recherche, production, purification et caracterisation de galactane-hydrolases pour la preparation des parois d'algues rouges", (February 1992)]. A detailed study of the .kappa.-carrageenase of Alteromonas carrageenovora was described by Potin et al. [Eur. J. Biochem. 228, 971-975 (1995)].
The availability of specific enzymes and tools for obtaining oligocarrageenans by genetic engineering could markedly improve their production.